The many distinct functions of actin in the cell are mediated by its interactions with numerous intracellular proteins. The studies proposed here focus on the three dimensional structure of the actin filament, the structures of several actin interacting proteins and specific interactions involved in actin's cellular functions. My approach, which involves the use of electron microscopy and image processing techniques, differs from previous studies of these proteins in that highly ordered forms of actin, with and without accessory proteins, have recently become available for such studies. Specifically, I propose to compare actin filament reconstructions from poly-lysine-induced paracrystals, Mg++ induced paracrystals and the acrosomal process from Limulus to obtain a consistent three-dimensional map of the actin filament to at least 2.5 nm resolution. This map will be used to align higher resolution models of the actin monomer, which have recently been obtained from crystalline actin sheets and from three-dimensional crystals, within the filament. Such alignment is necessary for understanding the actin monomer models in functional terms. Furthermore, I propose to localize functionally important sites on the actin molecule by stoichiometrically labelling crystalline actin sheets and/or paracrystals with actin interacting proteins. Stoichiometric binding of a given protein to crystalline actin sheets may 'force' the protein into an ordered arrangement in which it can be studied by image processing techniques. In addition, analysis of recently obtained 'tubes' formed from a stoichiometric complex of actin and DNase I will provide an example of an alternative method of mapping binding sites, as well as yielding structural information about the actin inter-acting molecule itself. I will attempt to use both labelling and 'cocrystallization' with a variety of actin-ligand combinations. Some actin interacting proteins are large enough that a single molecule or even its proteolytic fragments can be studied directly by electron microscopy. As an example of such a molecule, brain spectrin will be studied in terms of both its structure and its interaction with actin. This integrated approach should yield a higher resolution structural map of the actin filament overlaid with a functional map of biologically important binding sites, as well as structural information about interacting proteins that may mediate actin functions in vivo.